The cold-adapted γ-glutamyl-cysteine ligase from the psychrophile Pseudoalteromonas haloplanktis.

A recombinant γ-glutamyl-cysteine ligase from the psychrophile Pseudoalteromonas haloplanktis (rPhGshA II) was produced and characterised. This enzyme catalyses the first step of glutathione biosynthesis by forming γ-glutamyl-cysteine from glutamate and cysteine in an ATP-dependent reaction. The other ATP-dependent enzyme, glutathione synthetase (rPhGshB), involved in the second step of the biosynthesis, was already characterised. rPhGshA II is a monomer of 58 kDa and its activity was characterised through a direct radioisotopic method, measuring the rate of ATP hydrolysis. The enzyme was active even at cold temperatures in a moderately alkaline buffer containing a high concentration of Mg(++); 2-aminobutyrate could replace cysteine, although a lower activity was detected. The reaction rate of rPhGshA II at 15 °C was higher than that reported for rPhGshB, thus suggesting that formation of γ-glutamyl-cysteine was not the rate limiting step of glutathione biosynthesis in P. haloplanktis. rPhGshA II had different affinities for its substrates, as evaluated on the basis of the KM values for ATP (0.093 mM), glutamate (2.8 mM) and cysteine (0.050 mM). Reduced glutathione acted as an inhibitor of rPhGshA II, probably through the binding to an enzyme pocket different from the active site. Also the oxidised form of glutathione inhibited the enzyme with a more complex inhibition profile, due to the complete mono-glutathionylation of rPhGshA II on Cys 386, as proved by mass spectrometry data. When compared to rPhGshB, rPhGshA II possessed more typical features of a psychrophilic enzyme, as it was endowed with lower thermodependence and higher heat sensitivity. In conclusion, this work extends the knowledge on glutathione biosynthesis in the first cold-adapted source; however, another possible redundant γ-glutamyl-cysteine ligase (PhGshA I), not yet characterised, could participate in the biosynthesis of this cellular thiol in P. haloplanktis.

Properties of a putative cambialistic superoxide dismutase from the aerotolerant bacterium Streptococcus thermophilus strain LMG 18311.

The aerotolerance of the lactic - fermentative bacterium Streptococcus thermophilus is mainly based on the key antioxidant function of superoxide dismutase (StSOD). In this work, the comparison of recombinant StSOD (rStSOD) forms obtained from two different initiation triplets indicated that the enzyme from S. thermophilus strain LMG 18311 spans 201 residues. rStSOD is organised as a homodimer, even though protein aggregates are formed in concentrated solutions. The capability of binding and exchanging Fe or Mn in the active site classifies rStSOD as a putative cambialistic enzyme; the moderate preference for iron is counteracted by a 1.5-fold higher activity measured for the Mn-containing form. The enzyme is thermostable, being its half-inactivation time 10 min at 73.5°C; the energetic parameters of the heat inactivation process are regulated by the level of Mn cofactor. The effect of Mn content on the rStSOD sensitivity towards inhibitors and inactivators was also evaluated. Sodium azide acts as a weak inhibitor of rStSOD and its Mn content does not greatly affect this sensitivity. Concerning the physiological inactivator hydrogen peroxide, the Mn-enriched rStSOD displays a great resistance; a moderate sensitivity is instead observed in the presence of a low Mn content. Contrary to hydrogen peroxide, sodium peroxynitrite is a powerful inactivator, a behaviour enhanced in the Mn-enriched enzyme. All these results were compared with the corresponding data previously reported for the cambialistic SOD from the taxonomically related S. mutans. In S. thermophilus the regulation of the enzyme functions by the Mn content appears less relevant with respect to S. mutans.

Ofd1 controls dorso-ventral patterning and axoneme elongation during embryonic brain development.

Oral-facial-digital type I syndrome (OFDI) is a human X-linked dominant-male-lethal developmental disorder caused by mutations in the OFD1 gene. Similar to other inherited disorders associated to ciliary dysfunction OFD type I patients display neurological abnormalities. We characterized the neuronal phenotype that results from Ofd1 inactivation in early phases of mouse embryonic development and at post-natal stages. We determined that Ofd1 plays a crucial role in forebrain development, and in particular, in the control of dorso-ventral patterning and early corticogenesis. We observed abnormal activation of Sonic hedgehog (Shh), a major pathway modulating brain development. Ultrastructural studies demonstrated that early Ofd1 inactivation results in the absence of ciliary axonemes despite the presence of mature basal bodies that are correctly orientated and docked. Ofd1 inducible-mediated inactivation at birth does not affect ciliogenesis in the cortex, suggesting a developmental stage-dependent role for a basal body protein in ciliogenesis. Moreover, we showed defects in cytoskeletal organization and apical-basal polarity in Ofd1 mutant embryos, most likely due to lack of ciliary axonemes. Thus, the present study identifies Ofd1 as a developmental disease gene that is critical for forebrain development and ciliogenesis in embryonic life, and indicates that Ofd1 functions after docking and before elaboration of the axoneme in vivo.

Ofd1 is required in limb bud patterning and endochondral bone development.

Oral-facial-digital type I (OFDI) syndrome is an X-linked male lethal developmental disorder. It is ascribed to ciliary dysfunction and characterized by malformation of the face, oral cavity, and digits. Conditional inactivation using different Cre lines allowed us to study the role of the Ofd1 transcript in limb development. Immunofluorescence and ultrastructural studies showed that Ofd1 is necessary for correct ciliogenesis in the limb bud but not for cilia outgrowth, in contrast to what was previously shown for the embryonic node. Mutants with mesenchymal Ofd1 inactivation display severe polydactyly with loss of antero-posterior (A/P) digit patterning and shortened long bones. Loss of digit identity was found to be associated with a progressive loss of Shh signaling and an impaired processing of Gli3, whereas defects in limb outgrowth were due to defective Ihh signaling and to mineralization defects during endochondral bone formation. Our data demonstrate that Ofd1 plays a role in regulating digit number and identity during limb and skeletal patterning increasing insight on the functional role of primary cilia during development.

Regulation of the properties of superoxide dismutase from the dental pathogenic microorganism Streptococcus mutans by iron- and manganese-bound co-factor.

Streptococcus mutans, the main pathogen involved in the development of dental caries, is an aerotolerant microorganism. The bacterium lacks cytochromes and catalase, but possesses other antioxidant enzymes, such as superoxide dismutase (SmSOD). Previous researches suggested that SmSOD belongs to the 'cambialistic' group, functioning with Fe or Mn in the active site. A recombinant SmSOD (rSmSOD) with a His-tail has been produced and characterised. Studies on metal uptake and exchange proved that rSmSOD binds either Fe or Mn as a metal co-factor, even though with a consistent preference for Fe accommodation. The analysis of several enzyme samples with different values of the Mn/Fe ratio in the active site proved that the type of metal is crucial for the regulation of the activity of rSmSOD. Indeed, differently from the significant preference for Fe displayed by the enzyme in the binding reaction, its Mn-form was 71-fold more active compared to the Fe-form. The rSmSOD was endowed with a significant thermostability, its half-inactivation occurring after 10 min exposure at 71 or 73 degrees C, depending on the bound metal. Moreover, the enthalpic and entropic contribution to the heat inactivation process of rSmSOD were strongly regulated by the Mn content of the enzyme. The effect of typical inhibitors/inactivators has been investigated. rSmSOD was inhibited by sodium azide, and its sensitivity increased in the presence of higher Mn levels. Concerning two physiological inactivators, the enzyme displayed a different behaviour, being quite resistant to hydrogen peroxide and significantly sensitive to sodium peroxynitrite. Furthermore, the Mn co-factor had an amplifying role in the regulation of this different sensitivity. These results confirm the cambialistic nature of SmSOD and prove that its properties are regulated by the different metal content. The adaptative response of S. mutans during its aerobic exposure in the oral cavity could involve a different metal uptake by SmSOD.